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Zinc(II) Complexes of Ubiquitin: Speciation, Affinity and Binding Features
Author(s) -
Arena Giuseppe,
Fattorusso Roberto,
Grasso Giuseppe,
Grasso Giuseppa Ida,
Isernia Carla,
Malgieri Gaetano,
Milardi Danilo,
Rizzarelli Enrico
Publication year - 2011
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.201101364
Subject(s) - chemistry , zinc , ubiquitin , potentiometric titration , metal ions in aqueous solution , metal , titration , biophysics , ion , crystallography , inorganic chemistry , biochemistry , biology , organic chemistry , gene
Intraneuronal inclusions consisting of hypermetallated, (poly‐)ubiquitinated proteins are a hallmark of neurodegeneration. To highlight the possible role played by metal ions in the dysfunction of the ubiquitin–proteasome system, here we report on zinc(II)/ubiquitin binding in terms of affinity constants, speciation, preferential binding sites and effects on protein stability and self‐assembly. Potentiometric titrations allowed us to establish that at neutral pH only two species, ZnUb and Zn 2 Ub, are present in solution, in line with ESI‐MS data. A change in the diffusion coefficient of ubiquitin was observed by NMR DOSY experiments after addition of Zn II ions, and thus indicates metal‐promoted formation of protein assemblies. Analysis of 1 H, 15 N, 13 Cα and 13 CO chemical‐shift perturbation after equimolar addition of Zn II ions to ubiquitin outlined two different metal‐binding modes. The first involves a dynamic equilibrium in which zinc(II) is shared between a region including Met1, Gln2, Ile3, Phe4, Thr12, Leu15, Glu16, Val17, Glu18, Ile61 and Gln62 residues, which represent a site already described for copper binding, and a domain comprising Ile23, Glu24, Lys27, Ala28, Gln49, Glu51, Asp52, Arg54 and Thr55 residues. A second looser binding mode is centred on His68. Differential scanning calorimetry evidenced that addition of increasing amounts of Zn II ions does not affect protein thermal stability; rather it influences the shape of thermograms because of the increased propensity of ubiquitin to self‐associate. The results presented here indicate that Zn II ions may interact with specific regions of ubiquitin and promote protein–protein contacts.